Effect of chemotherapy on viability of Mycobacterium leprae as determined by ATP content, morphological index and FDA-EB fluorescent staining

Abstract

Viable bacterial populations were estimated in bacilli purified from 105 biopsies from 40 untreated and 65 multibacillary leprosy patients treated with multidrug therapy (MDT) for varying periods. The bacilli were purified and viability was determined by ATP content, morphological index (MI), and fluorescein diacetate-ethidium bromide (FDA-EB) staining. Viable populations were calculated, taking 3.58 x 10(-15) g/solid bacillus as the mean ATP content of a viable unit of Mycobacterium leprae. The proportion of viable bacilli was also estimated in the same specimens using solid-staining (MI) and green-staining bacilli by the FDA-EB method. In the untreated cases, the positive viability by ATP assay was 100%, 92% by MI, and 100% by FDA-EB. ATP content per solid bacillus was relatively constant, which was not the case with ATP content per green-staining bacillus. While the MI was zero in all cases, viable bacilli could still be detected by ATP estimations in 5 of the 32 (16%) patients after 2 years of MDT and in 1 of the 20 (5%) patients after 3 years of MDT. No viable bacilli could be detected even by this method beyond 3 years of MDT. On the other hand, green-staining bacilli were demonstrable in 7/32 (22%) of cases after 2 years of MDT, 2/20 (10%) after 3 years of MDT, and 1/13 (8%) after more than 3 years of treatment, indicating that the FDA-EB staining and ATP assay did not detect the same populations. A determination of the ATP content of M. leprae could be used as a reliable and sensitive tool for determining viability of the bacilli.